Basolateral amygdala bidirectionally modulates stressinduced hippocampal learning and memory deficits
through a p25/Cdk5-dependent pathway
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Citation
Rei, Damien, Xenos Mason, Jinsoo Seo, Johannes Gräff, Andrii
Rudenko, Jun Wang, Richard Rueda, et al. “Basolateral
Amygdala Bidirectionally Modulates Stress-Induced
Hippocampal Learning and Memory Deficits through a p25/Cdk5Dependent Pathway.” Proc Natl Acad Sci USA 112, no. 23 (May
20, 2015): 7291–7296.
As Published
http://dx.doi.org/10.1073/pnas.1415845112
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Thu May 26 03:55:10 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/100791
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Article is made available in accordance with the publisher's policy
and may be subject to US copyright law. Please refer to the
publisher's site for terms of use.
Detailed Terms
Basolateral amygdala bidirectionally modulates
stress-induced hippocampal learning and memory
deficits through a p25/Cdk5-dependent pathway
Damien Reia, Xenos Masona,1, Jinsoo Seoa,1, Johannes Gräffa,2, Andrii Rudenkoa, Jun Wanga, Richard Ruedaa,
Sandra Siegerta, Sukhee Choa, Rebecca G. Cantera, Alison E. Mungenasta, Karl Deisserothb,c, and Li-Huei Tsaia,3
a
Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
and bDepartment of Bioengineering and cHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305
Edited* by Ann M. Graybiel, Massachusetts Institute of Technology, Cambridge, MA, and approved March 24, 2015 (received for review August 15, 2014)
basolateral amygdala
p25/Cdk5 HDAC2
|
as well as hormonal cascades (15). Because of this complexity,
whether BLA activity affects hippocampus-dependent learning
and memory directly or indirectly through distinct relay brain regions or other downstream mediators, such as stress hormones,
remains unclear.
Cdk5 (cyclin-dependent kinase 5) plays a pleiotropic role in
the nervous system (16). This enzyme is essential for proper
brain development and regulates synaptic plasticity and cognitive function. Activation of Cdk5 requires association with a
regulatory subunit known as p35. p35 is subjected to calpainmediated cleavage into p25 in a process dependent on the
activation of glutamate receptors, specifically NR2B-containing NMDA receptors, following neurotoxic stimulation,
such as exposure to β-amyloid peptides, oxidative stress, or
Significance
Chronic stress has emerged in the epidemiologic literature as
a risk factor for both psychiatric and neurodegenerative
diseases. Thus, neurologic maladaptation to chronic stress is
highly relevant to the pathogenesis of human diseases such as
depression and Alzheimer’s disease, yet it remains poorly understood. Here we report a study of the neural circuits and
molecular pathways that govern the relationship between
stress and cognition. We present data demonstrating that behavioral stress impairs cognitive function via activation of a
specific direct neural circuit from the basolateral amygdala to
the dorsal hippocampus. Moreover, we delineate a molecular
mechanism by which behavioral stress is translated to hippocampal dysfunction via a p25/Cdk5 (cyclin-dependent kinase
5)-dependent pathway and epigenetic alterations of neuroplasticity-related gene expression.
| behavioral stress | cognitive dysfunction |
C
hronic stress can have devastating psychological consequences
that include depression and cognitive impairment (1–3).
Decades of research suggest that the hippocampus, a structure
important for learning and memory and implicated in depression,
is particularly sensitive to the effects of chronic stress. In animal
models, for example, chronic stress impairs hippocampus-dependent
forms of learning and memory (2). This sensitivity is partially
conferred by a dense concentration of glucocorticoid receptor
(GR) in the hippocampus (4), as well as through hippocampal
connectivity to important stress response coordinators, such as the
amygdala, from which the hippocampus receives abundant glutamatergic inputs (5–7). Following chronic stress, the hippocampus shows marked reductions in dendritic arborization and
neurogenesis, along with impaired plasticity (2). Many of these
effects have been attributed to connections between the hippocampus and a specific amygdalar subregion, the basolateral amygdala (BLA) (8–10).
Abundant evidence suggests that these BLA inputs have a major
impact on hippocampus function; for example, the hippocampus
and BLA synchronize their activity during fear memory retrieval
and fear extinction (11, 12), whereas electrical stimulation of the
BLA disrupts the induction of long-term potentiation (LTP), a
measure of synaptic plasticity, in the hippocampal CA1 subregion
(13). Lesions of the BLA have been shown to block the detrimental effects of repeated stress, a model of chronic stress in
rodents, on LTP and spatial memory (8, 10), as well as the
deleterious effect of hippocampal GR activation on hippocampus-dependent memory (9). Although the BLA sends abundant projections to the hippocampus (5–7), this region also
projects diffusely throughout the brain and thereby regulates a
myriad of behaviors, including valence or social interaction (14),
www.pnas.org/cgi/doi/10.1073/pnas.1415845112
Author contributions: This study was designed by D.R. and L.-H.T., and directed and coordinated by L.-H.T.; D.R. provided the samples and S.S. performed and analyzed the RTPCR experiment; K.D. provided the virus as well as expertise for the optogenetic experiments; D.R. performed all stereotaxic injections, behavior (except when otherwise
stated), and immunohistochemistry and mainly performed RFS, optogenetic stimulation,
and CNO treatment before RFS with the help of X.M.; all work on Δp35 mice was done by
D.R.; J.G. performed the Western blot analysis in Fig. 1 and novel object recognition
quantification in Figs. 1 and 3; novel location recognition was run by D.R. and X.M.
and quantified blindly by X.M.; J.S. performed hippocampal dissections for p25 Western
blot and Western blots; S.C. performed the electrophysiology analysis on the Gi-DREADD
BLA brain slices; immunohistochemistry analysis of HDAC2 and SYP were performed by
D.R., X.M., A.R., and R.R.; fiber optic placements were determined by X.M.; revisions were
performed by A.R., J.W., R.R., R.G.C., and A.E.M.; and the manuscript was written by D.R.,
X.M., A.E.M., and L.-H.T.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1
X.M. and J.S. contributed equally to this work.
2
Present address: Ecole Polytechnique Fédérale Lausanne, School of Life Sciences, Brain
Mind Institute, CH-1015 Lausanne, Switzerland.
3
To whom correspondence should be addressed. Email: lhtsai@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1415845112/-/DCSupplemental.
PNAS | June 9, 2015 | vol. 112 | no. 23 | 7291–7296
NEUROSCIENCE
Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the
hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by
generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25,
up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memoryrelated genes in the hippocampus. A combination of optogenetic
and pharmacosynthetic approaches shows that BLA activation is
both necessary and sufficient for stress-associated molecular
changes and memory impairments. Furthermore, we show that
this effect relies on direct glutamatergic projections from the
BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction.
Taken together, our data provide a neural circuit model for
stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation.
mice and found increased p25 levels compared with control
animals (Fig. 1B).
We next tested whether the increase in p25 generation following repeated stress was accompanied by an up-regulation of
HDAC2 and consequent decreases in memory-related genes (18,
19). These events are associated with p25 overexpression, decreases in learning and memory genes, and memory deficits in
neurotoxic conditions (18). We concentrated on the dorsal hippocampus and its CA1 subregion, because the dorsal hippocampus is involved in memory and spatial processing (31), and its
CA1 subregion is the output structure of the hippocampus and
is considered essential for novelty detection (32). We found that
RFS-treated animals exhibited increased HDAC2 immunoreactivity in the hippocampus. Furthermore, changes in HDAC2
were accompanied by decreased expression of known HDAC2regulated genes (19) in dorsal hippocampal CA1, including Synaptophysin (SYP, Fig. 1C), a presynaptic marker of functional
synapses, and Synapsin II (Syn. II) and Homer (Fig. S1C, Left and
Right, respectively), pre- and post-synaptic markers of functional
synapses, respectively. This stress paradigm was also shown to
decrease mRNA levels of other HDAC2-regulated genes (Fig.
S1D). A similar decrease in Synaptophysin and an increase in
HDAC2 were observed in dorsal hippocampal CA3 as well (Fig.
S1 E and F). This effect was confirmed using a restraint stress
paradigm (33). After 8 d of unpredictable daily restraint, generation of p25, HDAC2 up-regulation, and down-regulation of
Synaptophysin were also evident in hippocampal lysates from restrained animals compared with controls (Fig. S1G). In contrast,
RFS treatment did not alter p25 generation or HDAC2 and
Synaptophysin expression in the BLA compared with controls
(Fig. S1 H and I). This suggests that the observed changes are
brain region-specific.
Results
A Novel object
7292 | www.pnas.org/cgi/doi/10.1073/pnas.1415845112
D
CON
RFS
p35
p25
β-Tub.
HDAC2
*
RFS
HDAC2
SYP
0.8
0.4
0.0
3.0
pGR
2.0
GR
MERGED
1.5
**
1.0
0.5
0.0
SYP
1.6
1.2
Fold change
**
B
1.2
Fold change
0.0
CCON
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Fold change
0.6
0.5
0.4
0.3
0.2
0.1
**
Novel location
Fold change
ize the effects of stress on hippocampus-dependent learning and
memory pathways, we modified a learned-helplessness paradigm,
originally developed in rats (24), termed repetitive foot shock
(RFS), for use in Swiss Webster mice, a strain known for its
susceptibility to stress (25). This protocol is depicted in Fig. S1A
and described in SI Experimental Procedures.
Because different stress induction protocols have been shown
to either facilitate or impair learning and memory (3), we first
sought to establish the effects of our paradigm on hippocampusdependent tasks. To do this, we used two relatively low-stress
cognitive tests that have been validated in studies of hippocampal function (26): the novel object recognition task, which in
addition to the hippocampus also relies on perirhinal and prefrontal cortices (27), and novel location recognition (28). Detailed descriptions of these tasks can be found in SI Experimental
Procedures. During the first object training session, animals in
both the control and RFS-treated groups exhibited similar locomotor features, such as total distance moved and velocity, and
spent a comparable amount of time investigating the objects
(Fig. S1B). In these paradigms, control mice showed a significant
preference for the novel over the familiar object or location,
whereas RFS-treated mice performed no better than chance
(Fig. 1A). These data indicate that the RFS stress-induction
paradigm led to a deficit in hippocampus-dependent learning
and memory tasks, which is consistent with previous observations
suggesting that inescapable, uncontrollable repeated stress leads
to memory impairment (2, 8, 10).
To gain insight into the molecular mechanisms mediating the
deleterious effect of repeated stress on hippocampal function,
we examined the generation of p25 after RFS. We did so because
p25 is known to be generated by neuronal activity (29, 30), and
its sustained expression is known to be detrimental to learning and
memory (16). We analyzed the hippocampi of RFS-treated
0.8
0.7
Discrimination Index
Repeated Stress Leads to p25 Production, HDAC2 Elevation, and
Learning and Memory Deficits in the Hippocampus. To character-
Discrimination Index
excitotoxicity, as well as in response to physiological neuronal
activity (16).
A number of studies have implicated p25 production in
Alzheimer’s disease (AD)-like phenotypes, including learning
and memory impairments (16), and long-term overexpression
of p25 in the forebrain is known to lead to cognitive deficits (16).
Furthermore, stress and the heightened sensitivity to stress are
known risk factors for the development of AD (1). The role of p25
production after repeated stress remains undetermined, however.
One pathway through which p25/Cdk5 might be implicated in
stress-induced cognitive dysfunction is stress hormone receptormediated epigenetic signaling in the hippocampus. Indeed, it was
previously shown that GR is activated by p25/Cdk5-dependent
phosphorylation on Ser211 (17, 18), and that increased GR
phosphorylation leads to increased expression of histone deacetylase 2 (HDAC2) in a mouse model of AD (18). HDAC2 in turn
suppresses the expression of genes important for learning and
memory (18, 19), suggesting a mechanism by which elevated p25
generation leads to cognitive impairment. Although GR activation
has been shown to be required for stress-induced hippocampal
dysfunction and is dependent upon its phosphorylation (20–22), and
HDAC2 has been shown to be up-regulated in the ventral striatum
of mice following chronic stress (23), the possible up-regulation of
HDAC2 in the hippocampus after repeated stress, and the role of
p25/Cdk5 signaling in this process, are unknown. We tested the
hypothesis that p25 is generated in the hippocampus after repeated
stress in an amygdala-dependent manner and contributes to stressassociated learning and memory deficits. Blockade of p25 generation would then protect the hippocampus from the detrimental
effects of repeated stress.
Here we identify that the activity of a specific BLA to dorsal
hippocampus neural circuit mediates the detrimental effects of
repeated stress on hippocampal learning and memory via a
molecular pathway dependent on p25 generation.
0.8
0.4
**
0.0
GR
pGR
**
CON
RFS
*
1.0
0.0
Fig. 1. Repeated stress induces hippocampal molecular changes and learning
and memory deficits. (A) Effect of RFS treatment on learning and memory abilities
in the novel object recognition (n = 10 and 16) and novel location recognition
tasks (n = 10 per group; one-tailed t test). (B) Western blot images and quantification of the effect of repeated stress on p25 generation in the hippocampus
(n = 5 per group; unpaired t test). (C and D) Representative immunohistochemical
images and quantitative analysis of the effect of stress on HDAC2 and Synaptophysin (C) and GR and pGR (D) expression levels in the dorsal hippocampal
CA1 subregion (n = 4 per group; unpaired t test) (Synaptophysin and GR in
green; HDAC2 and pGR in red; DAPI in blue). Values are mean ± SEM. n.s.,
nonsignificant; P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (Scale bars: 20 μm.)
Rei et al.
BLA activation per se is sufficient to induce hippocampusrelated deficits in the absence of RFS, we expressed a channel
rhodopsin-2 (ChR2)-eYFP fusion protein or eYFP alone in BLA
pyramidal neurons (Fig. 3A and Fig. S3A, Left), combined with
implantation of a bilateral optical fiber over the BLA (Fig. 3A).
eYFP expression allowed us to identify the transduced BLA efferents at their destination in the dorsal hippocampus. Most of
the labeled afferents appeared to terminate in area CA3, with a
smaller proportion in CA1 (Fig. S3A). The dentate gyrus was
essentially devoid of any BLA efferents (not shown). We used an
8-d photostimulation protocol to mimic the RFS procedure (Fig.
S3B). This protocol was adapted from a previous study showing
that pairing the optogenetic activation of glutamatergic BLA
cells with a tone led to an association with fear learning (36).
Rei et al.
B
FP
FP i
eY N G N eY FS
-R
-CO -CO
4
Gi
FS
-R
Bilateral virus
injection
Fold change
A
p35
p25
FP
eY
AP: -1.34mm
eYFP-RFS
HDAC2
Gi-RFS
2.0
HDAC2
SYP
1.5
1.0
0.5
0.0
D
GR
5
pGR
GR
MERGED
4
3
2
1
0
*
***
- or
N
FS RFS
CO
Gi- YFP-R Gie
*
eYFP- or Gi-CON
eYFP-RFS
Gi-RFS
0.8
0.4
0.0
pGR
1
SYP
1.2
***
Fold change
eYFP-CON
**
2
0
β-Actin
C
3
E
Novel object
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
***
**
Novel location
0.7
Discrimination Index
Chronic BLA Cell Body Photostimulation Reproduces the Effect of
Repeated Stress on Learning and Memory. Next, to test whether
0.6
**
**
0.5
0.4
0.3
0.2
0.1
0.0
Fig. 2. BLA function is necessary for repeated stress-induced hippocampal
molecular changes and learning and memory deficits. (A) Schematic of the AAV5CaMKIIa-eYFP control and AAV5-CaMKIIa-HM4Di-IRES-mCitrine constructs and
mode of virus administration. (B–D) Effect of DREADD-induced BLA inhibition
during repeated stress on p25 generation (B) and of expression levels of
HDAC2 and Synaptophysin (C) and GR and pGR (D) in the dorsal hippocampal
CA1 subregion (n = 4, 5, and 6 per group, one-way ANOVA with Tukey’s post
hoc analysis) (pGR and HDAC2 in red; GR and Synaptophysin in green; DAPI
in blue). (E) Effect of DREADD-induced BLA inhibition on the performance of
RFS-treated mice in novel object recognition and novel location recognition
tasks (n = 10, 10, and 7). Values are mean ± SEM. n.s., nonsignificant; P > 0.05;
*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, one-tailed t test. (Scale bars: 20 μm.)
PNAS | June 9, 2015 | vol. 112 | no. 23 | 7293
NEUROSCIENCE
determine whether BLA projections to the hippocampus directly
mediate the effects of BLA activation on cognitive performance,
we conducted photostimulation of BLA axon terminals within
the hippocampus and asked whether this could recapitulate the
effects of RFS. Glutamatergic neurons of the BLA were transduced with AAV5-CaMKIIα-ChR2-eYFP or eYFP only. Unlike
before, optical fibers were implanted above either the dorsal
area or ventral area CA3 (Fig. S4A). These regions receive an
extensive network of projections from the BLA (Fig. S4B) and, in
the case of the dorsal hippocampus, are likely to activate BLA
fibers en route to the dorsal CA1 (5, 6). BLA terminals were
stimulated in the hippocampus using a protocol aimed at mimicking the RFS procedure (Fig. S4C).
We tested the efficiency of this procedure by measuring circulating corticosterone levels after stimulation, freezing levels
during light stimulation, and c-fos expression at the site of
stimulation (Fig. S5 A–C). Only photostimulation of the dorsal
hippocampus led to an increase in the number of c-fos–positive
cells in the dorsal hippocampus of ChR2 mice compared with
Discrimination Index
sought to map the upstream regulators implicated in the stressinduced impairment of hippocampal function. We examined
whether BLA activation is necessary for the effects of stress on the
hippocampus. We sought to chronically decrease the activity of
excitatory BLA neurons during RFS treatment using DREADD
(designer receptor exclusively activated by a designer drug)
technology (35). The inhibitory DREADD was transduced in
glutamatergic cells of the BLA using an adeno-associated virus
serotype 5 (AAV5) expressing the mutated Gi-coupled receptor
Gi-DREADD under control of the CaMKIIα promoter (AAV5CaMKIIa-HM4Di-IRES-mCitrine). Control animals received the
same virus expressing enhanced yellow fluorescent protein
(eYFP) only (AAV5-CaMKIIa-eYFP) (Fig. 2A). The experimental
procedure is depicted in Fig. S2A. The efficacy of the GiDREADD–mediated inhibition of BLA neurons was verified by
quantifying the effect of clozapine-N-oxide (CNO) treatment
upon c-fos expression in the transduced BLA cells, freezing levels
following fear conditioning, and by ex vivo slice recording (Fig.
S2 B–F and Table S1).
We then investigated the effect of BLA inhibition on hippocampal
learning and memory following repeated stress. Importantly, we
observed that BLA inactivation during RFS rescued the effects of
stress on both cognitive function and molecular pathology in the
hippocampus. Indeed, expression levels of p25, HDAC2, Synaptophysin, GR, and pGR were normalized in hippocampi of Gi-RFS
mice, as demonstrated by Western blot and immunohistochemistry
analyses (Fig. 2 B–D). The performance of Gi-RFS mice in novel
object recognition and novel location recognition tasks was also
indistinguishable from that of unstressed control mice (Fig. 2E).
These results suggest that the activity of glutamatergic neurons in
the BLA is necessary for stress-induced hippocampal dysfunction
and associated cognitive deficits.
Chronic Photostimulation of BLA Axon Terminals in the Dorsal Hippocampus Reproduces the Effect of Stress on Learning and Memory. To
Fold change
Pharmacosynthetic Inhibition of Glutamatergic Cells in the BLA Blocks
the Detrimental Effect of Stress on the Hippocampus. We next
Restricted expression in CamKII-positive cells and photostimulation
was verified by examining eYFP coexpression, c-fos expression,
and action potential firing in the BLA (Fig. S3 C–F), and the site
of implantation above the BLA was verified (Fig. S3G).
After repeated glutamatergic BLA cell body photostimulation,
we found increased p25 generation in the hippocampus (Fig. 3B),
increased HDAC2 expression, down-regulation of Synaptophysin
expression (Fig. 3C), and increased GR and pGR expression
(Fig. 3D) in the dorsal hippocampal CA1 subregion of ChR2 mice
compared with eYFP controls. Importantly, BLA activation also
impaired hippocampus-dependent memory formation, as measured
by novel object and novel location recognition tasks (Fig. 3E). These
results show that selective chronic activation of BLA glutamatergic
cell bodies is sufficient to reproduce molecular and behavioral effects previously associated with the RFS treatment.
Fold change
We next examined the expression levels of GR, which has a
low affinity for corticosterone and thus is typically activated only
after stress (4). GR can be activated via Cdk5-dependent phosphorylation on serine 211 (17) after behavioral stress (34) or neurotoxic stress (18). Samples from the dorsal CA1 of RFS-treated
animals exhibited an up-regulation in both protein expression and
phosphorylation of GR on Ser211 [phospho-GR (pGR)] compared
with controls (Fig. 1D). This indicates an increase in the total
amount of activated GR in dorsal CA1 neurons. After p25 generation, the Cdk5-mediated phosphorylation of GR has been shown
to lead to the up-regulation of HDAC2 and a subsequent downregulation of genes associated with learning and memory (18).
Our findings demonstrate that RFS treatment can serve as a
model of stress induction with reliable effects on hippocampal
function that can be measured using behavioral tests as well as
molecular indicators. Moreover, we describe the activation of an
RFS molecular pathway consisting of p25 generation; increased
GR, pGR, and HDAC2 expression; and decreases in HDAC2-regulated learning and memory genes in the hippocampus.
HDAC2
eYFP
ChR2
1.0
0.5
HDAC2
SYP
β-Actin 0.0
pGR
GR
1.5
pGR
***
E
Novel object
*
1.0
0.5
MERGED
0.0
0.8
0.7
0.5
*
0.6
0.4
0.2
Generation of p25 Is Necessary for the Detrimental Effect of Stress on
Learning and Memory. We directly examined the role of p25
0.0
0.0
Novel location
0.8
**
0.6
1.0
0.8
n.s.
0.5
0.4
0.3
0.2
0.1
0.7
generation in stress-induced learning and memory dysfunction by
taking advantage of a novel mouse model, the Δp35 knock-in
(KI) mouse (Δp35KI mice), in which p25 generation is abolished
(30) (Fig. S6A). Δp35KI and WT littermate mice were subjected
to RFS, as before. We found that RFS treatment did not result in
p25 generation in the Δp35KI hippocampus (Fig. S6B). Nor did
RFS affect Synaptophysin (Fig. 5A), GR, or pGR expression
levels in these mice (Fig. 5B). Additionally, HDAC2 up-regulation following stress was considerably reduced in the p35KI mice
compared to control littermates (Fig. 5A). Remarkably, these
mice also appeared to be resilient to RFS-induced impairments in hippocampus-dependent memory formation, with the
performance of RFS-treated Δp35KI mice indistinguishable
from that of unstressed controls (Fig. 5C). These in vivo experiments suggest that production of p25 in the hippocampus is
necessary for the behavioral and molecular phenotypes that
manifest in the hippocampus following repeated stress.
eYFP
ChR2
***
n.s.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0
Fig. 3. BLA activation is sufficient to reproduce repeated stress-induced
hippocampal molecular changes and learning and memory deficits.
(A) Schematic of the AAV5-CaMKIIa-eYFP and AAV5-CaMKIIa-ChR2-eYFP
constructs, mode of virus administration, and fiber optic placement. (B) Effect
of BLA photostimulation on p25 generation in the hippocampus (n = 5 per
group; unpaired t test). (C and D) Representative immunohistochemical images
and quantitative analysis of HDAC2 and Synaptophysin (C) and GR and pGR
(D) expression levels in the dorsal hippocampal CA1 subregion from eYFP and
ChR2 mice (n = 5 per group; unpaired t test) (HDAC2 and pGR in red; Synaptophysin and GR in green; DAPI in blue). (E) Effect of optogenetic BLA
activation on performance in novel object recognition and novel location
recognition behavioral tasks (n = 10 CON mice and 7 ChR2 mice; one-tailed
t test). Values are mean ± SEM. n.s., nonsignificant; P > 0.05; *P ≤ 0.05; **P ≤
0.01; ***P ≤ 0.001. (Scale bars: 20 μm.)
Discussion
The data presented here demonstrate that repeated stress activates a molecular pathway in the hippocampus consisting of p25
generation, GR up-regulation and phosphorylation, and HDAC2
up-regulation. These phenotypes are accompanied by the downregulation of memory-related markers in the hippocampus and
impairments of learning and memory. We found that this pathway is activated by direct glutamatergic projections from the BLA
to the dorsal hippocampus, and that these phenotypes are rescued
in the absence of p25 generation. This work details the mechanisms
of how repeated stress impacts hippocampus-associated learning
and memory at the neural circuit and molecular levels (see the
proposed model in Fig. S7).
control mice (Fig. S5C), consistent with the notion that the two
hippocampal domains act independently of each other (31).
Photostimulation of the BLA inputs to the dorsal hippocampus
(Fig. 4A), but not the ventral hippocampus (Fig. S5D), increased
p25 generation in ChR2-expressing mice compared with eYFP
controls. Dorsal, but not ventral, photostimulation of BLA terminals was also associated with increased immunoreactivity for
HDAC2, reduced expression of Synaptophysin, and increased
GR and pGR expression in the dorsal CA1 of ChR2 mice
compared to eYFP controls (Fig. 4 B and C and Fig. S5 E and F).
In the case of the dorsally stimulated mice, this was accompanied
by an impaired performance in both the novel object and novel
location recognition tasks (Fig. 4D). In contrast, illumination of the
ventral hippocampus in ChR2-transduced animals impaired performance in the novel object recognition task only (Fig. S5G).
HDAC2
ChR2-Dorsal
p25
2.0
p35
p25
β-Tubulin
CeYFP-Dorsal
studies have concluded that BLA stimulation leads to LTP deficits in hippocampal CA1, and that the BLA is necessary for the
detrimental effects of chronic stress on spatial memory (8, 10,
BeYFP-Dorsal
ChR2Dorsal
*
1.5
1.0
0.5
HDAC2
SYP
0.0
ChR2-Dorsal
2.0
pGR
GR
MERGED
Fold change
Dorsal
Fold change
A eYFP-
Modulation of Hippocampal Function by the BLA and the Importance
of the Direct BLA to Dorsal Hippocampus Connections. Previous
1.5
1.0
0.5
0.0
GR
pGR
*
*
D
Novel object
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
***
n.s.
Fold change
2.0
GR
Discrimination Index
ChR2
Fold change
DeYFP
AP: -1.34mm
Discrimination Index
p25
**
1.5
Fold change
*
Fold change
1.5
p35
Fold change
eYFP ChR2
Bilateral virus injection
Laser stimulation
These results suggest that selective activation of glutamatergic
projections from the BLA primarily to the dorsal, but not the
ventral, hippocampus reproduced the effects of repeated stress
on the hippocampal CA1 subregion and behavioral measures of
learning and memory.
SYP
1.0
2.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
SYP
1.2
**
Fold change
C
1.0
0.8
**
0.6
0.4
0.2
0.0
Novel location
Discrimination Index
B
Discrimination Index
A
0.7
0.6
0.5
***
n.s.
eYFP
ChR2-Dorsal
0.4
0.3
0.2
0.1
0.0
Fig. 4. Activation of direct projections from the BLA to the dorsal hippocampus is sufficient to reproduce repeated stress-induced hippocampal molecular
changes and learning and memory deficits. (A) Effect of BLA terminal photostimulation on p25 expression level, measured by Western blot analysis from
hippocampal lysates (unpaired t test). (B and C) Representative immunohistochemical images and quantitative analysis of HDAC2 and Synaptophysin (B) and
GR and pGR (C) expression levels in dorsal CA1 after BLA terminal photostimulation in the dorsal hippocampus (n = 5 per group; unpaired t test for HDAC2
and Synaptophysin, one-way ANOVA with Tukey’s post hoc analysis for GR/pGR) (pGR and HDAC2 in red; GR and Synaptophysin in green; DAPI in blue).
(D) Effect of dorsal BLA terminal photostimulation on novel object (NO) and novel location (NL) recognition behavioral tasks (one-tailed t test). n = 16 for
eYFP and 10 for ChR2. Values are mean ± SEM. n.s., nonsignificant; P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (Scale bars: 20 μm.)
7294 | www.pnas.org/cgi/doi/10.1073/pnas.1415845112
Rei et al.
WT-CON
WT-RFS
∆p35-CON
∆p35-RFS
HDAC2
**
n.s.
***
2.0
SYP
n.s.
1.2
1.0
0.5
GR
pGR
*
**
n.s.
pGR
GR
MERGED
Fold change
2.5
2.0
1.5
1.0
0.5
0.0
WT-CON
WT-RFS
∆p35-CON
∆p35-RFS
0.8
0.6
0.4
0.2
0.0
0.0
B
Fold change
1.5
*
1.0
n.s.
C
Discrimination Index
HDAC2
SYP
Fold change
2.5
Novel object Novel location
0.8
0.7
* **
**
0.6
0.5
n.s.
0.4
0.3
0.2
0.1
0.0
0.8
Discrimination Index
A
0.7
*
**
**
0.6
0.5
n.s.
0.4
0.3
0.2
0.1
0.0
Fig. 5. p25 generation is necessary for repeated stress-induced hippocampal molecular changes and learning and memory deficits. (A and B) Absence of RFSinduced changes in HDAC2 and Synaptophysin (A) and GR and pGR (B) expression levels in the hippocampal CA1 subregion in the Δp35KI hippocampus (n = 4
per group; one-way ANOVA Tukey’s post hoc analysis) (HDAC2 and pGR in red; Synaptophysin and GR in green; DAPI in blue). (C) Absence of repeated stressinduced learning and memory deficits in the Δp35KI mouse. (n = 8 for WT CON and RFS; n = 10 and 11 for Δp35 CON and RFS, respectively; one-sample onetailed t test). Values are mean ± SEM. n.s., nonsignificant; P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (Scale bars: 20 μm.)
Subregion-Specific Effects of BLA Inputs into the Hippocampus. We
were surprised to find that the specific activation of BLA inputs
into the dorsal, but not the ventral, hippocampus recapitulated
the effect of repeated stress on hippocampal function. Functional differences between hippocampal subdomains have previously been suggested by the finding of a higher density of place
cells in the dorsal hippocampus compared with the ventral
hippocampus, which provides finer spatial tuning (31). In addition, the dorsal CA1 area is thought to be essential for novelty
detection (32, 38). This could explain why the novel location
recognition task was unaffected in the ventrally stimulated
animals. These two hippocampal regions also exhibit a number of
differences in their connectivity patterns; for example, dorsal
CA1 has extensive reciprocal connections to association cortices,
whereas ventral CA1 has a greater degree of connectivity to
subcortical areas, such as the hypothalamus and amygdala (39).
Accordingly, lesions of the dorsal hippocampus impair memory
Rei et al.
and spatial processing, whereas lesions of the ventral hippocampus
impair emotional, social, and endocrine regulation (31). Patterns of
corticosteroid receptor expression also differ between these regions,
with a higher level of GR expression in the dorsal CA1 (4). Perhaps
a higher GR tone in this region can amplify input from the BLA,
resulting in greater sensitivity to glutamate and p25 generation
compared with that in the ventral region.
p25 Generation Is Necessary for the Negative Effects of Repeated Stress
on the Hippocampus and Is Modulated at the Neural Circuit Level. We
have shown that the activity of specific BLA inputs leads to
p25 generation and increased HDAC2 expression in the dorsal
hippocampus and that p25 generation is necessary to induce
hippocampus-dependent learning and memory deficits following repeated stress. The pathway from p25 generation to
HDAC2-associated decreases in learning and memory genes,
which lead to cognitive impairment, has been demonstrated
previously (18, 19). The present study shows that p25 generation links the activation of a specific neural circuit following
stress with epigenetic changes associated with learning and
memory impairment.
Tracing studies have uncovered a highly conserved amygdalohippocampal circuitry in rodents and nonhuman primates that
likely is similar in humans (5, 40). This conservation raises the
potential that specific therapies aimed at restraining the activity
of the BLA to inhibit p25 generation, or to reduce the associated Cdk5 overactivation, may effectively alleviate cognitive
symptoms in the host of neurologic, psychiatric, and systemic
diseases for which stress is emerging as both a causative and
exacerbating factor.
Experimental Procedures
All mouse work was approved by the Committee for Animal Care of the
Division of Comparative Medicine at Massachusetts Institute of Technology.
RFS procedure consisted of submitting the mice to the delivery of 10 foot
shocks at random intervals during an hour, daily for 8 d. DREADD-BLA inhibition was induced during the RFS paradigm. Photostimulation of the BLA
or its fibers in the hippocampus was made at 20 Hz for 2 or 20 s, respectively,
and repeated 10 times daily for 8 d. Detailed information on materials and
methods, including information on animals and the RFS paradigm, behavioral
assays, Western blot analysis, immunohistochemistry, qRT-PCR, Gi-DREADD,
stereotaxic ChR2 injection and optical fiber placement, optogenetic stimulation, corticosterone assays, generation of the Δp35KI mice, and statistical
analysis, is provided in SI Experimental Procedures.
ACKNOWLEDGMENTS. We thank Alexander Nott, Adam Bero, Susan Su, and
Kay Tye for their careful reading of the manuscript. This study was supported
by National Institutes of Health Grants AG047661 and NS051874, and funding
from the JPB Foundation (to L.-H.T.). X.M. was a 2012–2013 Howard Hughes
Medical Institute Medical Research Fellow. J.G. was supported by a Bard Richmond Fellowship and a Swiss National Science Foundation grant for prospective
PNAS | June 9, 2015 | vol. 112 | no. 23 | 7295
NEUROSCIENCE
13). In the present study, we used genetic and pharmacosynthetic
methods to examine the function of the BLA in a paradigm
reminiscent of repeated stress and its impact on cognition, with a
degree of cell- and circuit-specific modulation not attainable by
previous electric stimulation and pharmacologic or physical cell
inactivation paradigms (8, 13, 37). Here we describe the pivotal
role of glutamatergic cell activity in the BLA to modulate the
stress-related hippocampal phenotypes. We show that their activation is necessary for the detrimental effect of repeated stress
on hippocampal-associated learning and memory, and that repeated
optogenetic activation of those cells (in the absence of a stressor) is
sufficient to reproduce the effects of repeated stress on the hippocampus. Taking advantage of the anterograde transport of ChR2
along the axons of BLA neurons to the hippocampus, we were able
to show via terminal photostimulation that the effects of BLA activation on the hippocampus are mediated directly, as opposed to
being reliant on an intermediate structure or circulating hormones.
Consistent with this, we found that BLA terminal stimulation in the
dorsal hippocampus and ventral hippocampus led to an increase in
circulating levels of corticosterone, whereas only the former recapitulated the effect of stress on the hippocampus. This is consistent with the notion that changes in hippocampal function affect
glucocorticoid secretion (15). Furthermore, the fact that ventral
stimulation induced an increase in corticosterone without fully affecting hippocampal function confirms earlier work showing that
the increase in circulating corticosterone levels alone is insufficient
to induce hippocampal dysfunction in the absence of a functional
amygdala (9, 22). We now show that this requirement is due to the
necessary BLA input onto the dorsal hippocampus. The question of
whether or not corticosterone is a necessary or permissive factor in
the impact of this circuit on hippocampal function remains to be
formally addressed.
researchers. S.S. was supported by a Human Frontier Science Program long-term
postdoctoral fellowship. K.D. is an investigator at the Howard Hughes Medical
Institute and the National Institutes of Health. L.-H.T. is an investigator at the
Picower Institute for Learning and Memory.
1. Aznar S, Knudsen GM (2011) Depression and Alzheimer’s disease: Is stress the initiating factor in a common neuropathological cascade? J Alzheimers Dis 23(2):
177–193.
2. Sandi C, Pinelo-Nava MT (2007) Stress and memory: Behavioral effects and neurobiological mechanisms. Neural plasticity 2007:78970.
3. Lupien SJ, McEwen BS, Gunnar MR, Heim C (2009) Effects of stress throughout the
lifespan on the brain, behaviour and cognition. Nat Rev Neurosci 10(6):434–445.
4. Reul JM, de Kloet ER (1985) Two receptor systems for corticosterone in rat brain:
Microdistribution and differential occupation. Endocrinology 117(6):2505–2511.
5. Pikkarainen M, Ronkko S, Savander V, Insausti R, Pitkanen A (1999) Projections from
the lateral, basal, and accessory basal nuclei of the amygdala to the hippocampal
formation in rat. J Comp Neurol 403(2):229–260.
6. Pitkanen A, Pikkarainen M, Nurminen N, Ylinen A (2000) Reciprocal connections between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A review. Ann N Y Acad Sci 911:369–391.
7. Felix-Ortiz AC, et al. (2013) BLA to vHPC inputs modulate anxiety-related behaviors.
Neuron 79(4):658–664.
8. Kim JJ, Koo JW, Lee HJ, Han JS (2005) Amygdalar inactivation blocks stress-induced
impairments in hippocampal long-term potentiation and spatial memory. J Neurosci
25(6):1532–1539.
9. Roozendaal B, McEwen BS, Chattarji S (2009) Stress, memory and the amygdala. Nat
Rev Neurosci 10(6):423–433.
10. Kim JJ, Lee HJ, Han JS, Packard MG (2001) Amygdala is critical for stress-induced modulation of hippocampal long-term potentiation and learning. J Neurosci 21(14):5222–5228.
11. Seidenbecher T, Laxmi TR, Stork O, Pape HC (2003) Amygdalar and hippocampal theta
rhythm synchronization during fear memory retrieval. Science 301(5634):846–850.
12. Lesting J, et al. (2011) Patterns of coupled theta activity in amygdala-hippocampalprefrontal cortical circuits during fear extinction. PLoS One 6(6):e21714.
13. Vouimba RM, Richter-Levin G (2005) Physiological dissociation in hippocampal subregions in response to amygdala stimulation. Cereb Cortex 15(11):1815–1821.
14. Fernando AB, Murray JE, Milton AL (2013) The amygdala: Securing pleasure and
avoiding pain. Front Behav Neurosci 7:190.
15. Herman JP, Ostrander MM, Mueller NK, Figueiredo H (2005) Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis. Prog Neuropsychopharmacol Biol Psychiatry 29(8):1201–1213.
16. Su SC, Tsai LH (2011) Cyclin-dependent kinases in brain development and disease.
Annu Rev Cell Dev Biol 27:465–491.
17. Kino T, et al. (2007) Cyclin-dependent kinase 5 differentially regulates the transcriptional activity of the glucocorticoid receptor through phosphorylation: Clinical implications for the nervous system response to glucocorticoids and stress. Mol
Endocrinol 21(7):1552–1568.
18. Gräff J, et al. (2012) An epigenetic blockade of cognitive functions in the neurodegenerating brain. Nature 483(7388):222–226.
19. Guan JS, et al. (2009) HDAC2 negatively regulates memory formation and synaptic
plasticity. Nature 459(7243):55–60.
20. Kamal A, Ramakers GM, Altinbilek B, Kas MJ (2014) Social isolation stress reduces
hippocampal long-term potentiation: Effect of animal strain and involvement of
glucocorticoid receptors. Neuroscience 256:262–270.
21. Kim JJ, Diamond DM (2002) The stressed hippocampus, synaptic plasticity and lost
memories. Nat Rev Neurosci 3(6):453–462.
22. Krugers HJ, Goltstein PM, van der Linden S, Joels M (2006) Blockade of glucocorticoid
receptors rapidly restores hippocampal CA1 synaptic plasticity after exposure to
chronic stress. Eur J Neurosci 23(11):3051–3055.
23. Uchida S, et al. (2011) Epigenetic status of Gdnf in the ventral striatum determines
susceptibility and adaptation to daily stressful events. Neuron 69(2):359–372.
24. Grahn RE, Watkins LR, Maier SF (2000) Impaired escape performance and enhanced conditioned fear in rats following exposure to an uncontrollable stressor
are mediated by glutamate and nitric oxide in the dorsal raphe nucleus. Behav Brain
Res 112(1-2):33–41.
25. Adamec R, Walling S, Burton P (2004) Long-lasting, selective, anxiogenic effects of
feline predator stress in mice. Physiol Behav 83(3):401–410.
26. Manns JR, Eichenbaum H (2009) A cognitive map for object memory in the hippocampus. Learn Mem 16(10):616–624.
27. Broadbent NJ, Gaskin S, Squire LR, Clark RE (2010) Object recognition memory and
the rodent hippocampus. Learn Mem 17(1):5–11.
28. Mumby DG, Gaskin S, Glenn MJ, Schramek TE, Lehmann H (2002) Hippocampal
damage and exploratory preferences in rats: Memory for objects, places, and contexts. Learn Mem 9(2):49–57.
29. Engmann O, et al. (2011) Cyclin-dependent kinase 5 activator p25 is generated during
memory formation and is reduced at an early stage in Alzheimer’s disease. Biol Psychiatry 70(2):159–168.
30. Seo J, et al. (2014) Activity-dependent p25 generation regulates synaptic plasticity
and Aβ-induced cognitive impairment. Cell 157(2):486–498.
31. Fanselow MS, Dong HW (2010) Are the dorsal and ventral hippocampus functionally
distinct structures? Neuron 65(1):7–19.
32. Lisman JE, Otmakhova NA (2001) Storage, recall, and novelty detection of sequences
by the hippocampus: Elaborating on the SOCRATIC model to account for normal and
aberrant effects of dopamine. Hippocampus 11(5):551–568.
33. Buynitsky T, Mostofsky DI (2009) Restraint stress in biobehavioral research: Recent
developments. Neurosci Biobehav Rev 33(7):1089–1098.
34. Adzic M, et al. (2009) Acute or chronic stress induce cell compartment-specific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in Wistar
rat brain. J Endocrinol 202(1):87–97.
35. Sternson SM, Roth BL (2014) Chemogenetic tools to interrogate brain functions. Annu
Rev Neurosci 37:387–407.
36. Johansen JP, et al. (2010) Optical activation of lateral amygdala pyramidal cells instructs associative fear learning. Proc Natl Acad Sci USA 107(28):12692–12697.
37. Kim EJ, Kim ES, Park M, Cho J, Kim JJ (2012) Amygdalar stimulation produces alterations on firing properties of hippocampal place cells. J Neurosci 32(33):11424–11434.
38. Blum KI, Abbott LF (1996) A model of spatial map formation in the hippocampus of
the rat. Neural Comput 8(1):85–93.
39. Naber PA, Witter MP (1998) Subicular efferents are organized mostly as parallel
projections: a double-labeling, retrograde-tracing study in the rat. J Comp Neurol
393(3):284–297.
40. Aggleton JP (1986) A description of the amygdalo-hippocampal interconnections in
the macaque monkey. Exp Brain Res 64(3):515–526.
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Rei et al.